Microbial protein expression system

ABSTRACT

The present invention provides bacterial strains for secretion of soluble biologically active recombinant heterologous proteins into periplasm or on a surface/into a cultivation medium of bacteria. The invention exploits the secretion system of Gram-negative bacteria including periplasmic chaperones and usher/secretin proteins. For accomplishing the aim, the bacterial strains simultaneously express the fusion protein (signal peptide)-(mature hecterologous protein)-(subunit of a bacterial surface structure, Caf1), periplasmic chaperone specific for the subunit, and outer membrane usher/secretin protein specific for the subunit. Secretion of fusion proteins: (signal peptide of Caf1)-(mature human IL-1β)-(mature Caf1), (signal peptide of Caf1)-(mature human GM-CSF)-(mature Caf1), and (signal peptide of Caf1)-(mature human IL-1ra)-(mature Caf1) that were expressed in  Escherichia coli  simultaneously with the periplasmic chaperone Caf1M and the usher/secretin protein Caf1A are examples of the use of the invention.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/FI00/00387 which has an Internationalfiling date of May 3, 2000, which designated the United States ofAmerica and was published in English.

FIELD OF INVENTION

This invention is related to biotechnology and more specifically toproduction of recombinant heterologous proteins by microbes. Inparticular, this invention concerns secretion of soluble biologicallyactive heterologous proteins into periplasm and/or on a surface/into acultivation medium of Gram-negative bacteria. The invention exploits thesecretion system of Gram-negative bacteria including periplasmicchaperones and usher/secretin proteins of the system.

BACKGROUND OF INVENTION

Commercial production of various medically and industrially valuablerecombinant proteins by microbes is one the key challenges of modembiotechnology. Even though such systems are known, there are severetechnical problems which are encountered within large-scale exploitationof microbial cell machinery. There are several secretion systems inGram-negative bacteria which can be potentially exploited for secretionof recombinant heterologous proteins. The systems are briefly reviewedbelow.

1. Secretion Across the Inner Membrane

The majority of secreted proteins in Escherichia coli are synthesized asprecursors with a classic N-terminal signal peptide (SP) which isessential for efficient export and which is cleaved during or followingtranslocation across the inner membrane. Translocation is mediated bythe Sec translocase (SecA/Y/G/E). SecA is a peripherally associatedATPase, which interacts with the signal sequence and mature part of theprecursor, guides the polypeptide into the translocator and providesenergy for the process. Sec Y, E and G are integral membrane proteinsthat form the translocator itself and a central aqueous channel throughwhich the polypeptide is translocated. SecD and F have large periplasmicdomains. Proposed functions of these two proteins are related to latersteps in the process and have included release from the membrane andmediation of transfer of energy from the proton motive force.Polypeptides are translocated in an ‘unfolded’ state. Hence SecB is acytosolic chaperone apparently dedicated to the secretion pathway whichis required for export of a subset of secreted proteins. SecB bothinhibits premature folding and targets the precursor to the membranetranslocase complex. It now appears that basic principles of the exportsystem are universal. Although translocation is primarilyco-translational in eukaryotic systems and targeting to the secretionapparatus is primarily via SRP (signal recognition particle), the coretranslocator is homologous in both systems (yeast Sec 61 α and γ arehomologous of E. coli SecY/E). Also, E. coli ffh and 4.5S RNA arehomologous of eukaryotic 54 KD subunit and 7S RNA of SRP, respectively.Comparison of the eukaryotic and prokaryotic systems has beenextensively reviewed recently (Rapoport, T., et al. (1996)Annual Reviewof Biochemistry. 65:271-303; Schatz, G., and B. Dobberstein(1996)Science. 271:1519-1526).

Similarities in the basic function of eukaryotic and bacterial exportsystems have meant that some mammalian proteins can be successfullysecreted to the periplasm of E. coli. Examples include human insulin.Often, however, fine tuning is required such as optimising theN-terminus of the mature protein, removal of positively charged residuesat the end of the SP or beginning of the mature protein, ensuringpresence of a good cleavage site. Frequently, a bacterial signal peptidesuch as the OmpA SP has been used. The Caf1 signal sequence has alsobeen successfully used to export mammalian cytokines (see below). Amajor problem on recombinant expression in E. coli is incorrect foldingwith accompanying protein degradation or accumulation in an insolubleand inactive form as inclusion bodies.

In addition to the sec dependent secretion system there are at least twoother systems of protein translocation across the bacterial innermembrane. The M13 phage coat protein is also synthesised with anadditional SP, but assembly of this protein across the membrane isindependent of the sec machinery. Recently, a novel pathway involved insecretion of cofactor-containing proteins has been elucidated (Santini,G., et al. (1998) EMBO Journal, 17:101-112; Weiner, J., et al. (1998)Cell. 93:93-101). Proteins following this pathway have a long leadercontaining a characteristic ‘twin arginine’ motif. It is proposed thatcofactor attachment occurs in the cytosol and that the fully foldedprotein is translocated across the inner membrane via products of themttABC operon. In addition, there are cytosolic proteins of E. coliwhich appear to be localised in a priviledged site which is sensitive toosmotic shock. Therefore may have some transient access to theperiplasm. Such proteins include thioredoxin (Lunn, C. A., and V. P.Pigiet. (1982) J.Biol.Chem. 257:11424-11430) involved in disulphidereduction of cellular components, the cytosolic chaperone DnaK(Yaagoubi, A.,et al. (1994) Journal of Bacteriology. 176:7074-7078),elongation factor Tu (Jacobson, G., et al. (1976) Biochemistry.15:2297-2303) and inner membrane bound components of enterobactinsynthase complex (Hantash, F., et al. (1997) Microbiology. 143:147-156),and capsule assembly (Rigg, G., et al. (1998) Microbiology.144:2905-2914).

It has been suggested that this ‘compartment’ may be related totransient formation of adhesion zones between the bacterial inner andouter membranes, but nothing is known regarding properties of theprotein which targets them to this location nor about the physicalnature of this ‘compartment’. A number of cytosolic recombinant proteins(i.e. without SP) also behave in a similar manner and are thuspresumably targeted to the same cellular location. These include GSTfusion proteins, interleukin 1β (Joseph-Liauzun, E., et al. (1990) Gene.86:291-295).

2. Extracellular Secretion Systems

Six different pathways for export of extracellular proteins have beenidentified in Gram negative bacteria. Each pathway has been identifiedin a diverse range of bacteria. The basic properties of these systemsare summarised in FIG. 1 (recently reviewed by Lory (Lory, S. (1998)Current Opinion in Microbiology. 1:27-35).

The Type II pathway, which is considered to be the main terminal branchof the sec- dependent pathway, is used for export of many differentunrelated soluble proteins. It involves a folded periplasmicintermediate and requires approximately 12 dedicated genes for exportacross the OM. Alternative terminal branches to the sec pathway includespecific chaperone-dependent fimbriae assembly and the OM helperpathway. The former pathway also involves a periplasmic intermediate (atleast partially folded) but in this case the secreted polypeptide isspecifically transported in association with its own chaperone/outermembrane usher protein system. Outer membrane helpers fold into theouter membrane with concomitant exposure of the effector domain at thecell surface and, in the case of IgA protease, release viaself-hydrolysis. Interaction with general periplasmic chaperones e.g.DsbA has been demonstrated as a critical step in secretion pathway for anumber of sec dependent proteins.

Type I, Type III, and most members of Type IV pathways are secindependent and mediate secretion of a specific protein (subset ofproteins or DNA (Type IV) directly from the cytosol. Type I results insecretion into the external media, whereas Type III targets the secretedprotein directly into the eukaryotic cell following contact-stimulatedactivation of the secretion system. The Type III pathway also sharesmany features with flagellar assembly systems.

3. Secretion of Recombinant Heterologous Proteins in Gram-negativeBacteria

Incorrect folding of proteins in the cytosol may lead to degradation orformation of misfolded protein as inclusion bodies. In many instances,therefore, it is desirable to have heterologous expression ofrecombinant proteins in the bacterial periplasm, at the cell surface, orin the extracellular media, permitting correct folding and formation ofa functional product. Proteins secreted to the periplasm of E. coli arein an oxidising environment, compared to the reducing environment of thecytosol. The periplasm contains oxidoreductases and chaperones(disulphide bond isomerase, DsbA and C, peptidyl prolylcis-transisomerase, RotA. SurA, and FkpA) which are essential for thecorrect folding of proteins (Missiakas. D., and S.Raina. (1997) Journalof Bacteriology 179:2465-2471). In addition, recombinant proteinsexpressed in the periplasm or secreted to the extracellular medium wouldrepresent a high percentage of the final protein content of theserespective compartments. Thus, when the final goal is to obtain apurified recombinant product, secretion of the product to the periplasmor externally should greatly facilitate purification protocols. Althoughthere are quite a few systems available for periplasmic localisation ofproteins, there is no major system for secretion of extracellularproducts from E. coli. Over the past decade there has also been a greatdeal of interest in expressing proteins and peptides on the surface ofmicroorganisms. Phage display technology (Winter, G., et al. (1994)Annual Review of Immunology 12:433-455) utilises the coat protein offilamentous bacteriophage for surface display of proteins or peptides.Such technology has been applied to the isolation of specific antibodyfragments and for the rapid identification of peptide ligands. Interestin surface display in E. coli (Georgiou, G., et al. (1993) Trends inBiotechnology. 11:6-10) and other Gram negative bacteria has centeredaround identification of protective epitopes and their applications aslive vaccines, production of bacterial adsorbents and whole-cellbiocatalysts.

Although there has been some success in expressing of proteins, thereare a number of limitations within the existing systems as outlinedbelow.

Most secretory/assembly pathways of E. coli have been investigated fortheir potential exploitation as secretion vehicles for heterologousproteins. These include systems that direct the protein to theperiplasm, cell surface or extracellular medium.

3.1 SP Alone

A number of expression vectors use a bacterial SP (often that of the OMprotein OmpA) to mediate export across the inner membrane. Destinationof the protein depends on the nature of the protein itself. It is notuncommon for proteins exported in this way in high levels to forminsoluble complexes, inclusion bodies, in the periplasm as a result ofincomplete folding.

3.2. Affinity Purification Systems

Fusion expression systems have been developed to facilitate downstreampurification of recombinant products. Examples include insertion of aHis tag for purification on a Nickel column (Clontech, Qiagen, Invitrogen); fusion to MalE (New England Biolabs), maltose bindingprotein, with subsequent purification on an amylose column; thioredoxinfusions with PAO (phenyl arsine oxide) resin and chitin binding domainfusions with chitin columns (New England Biolabs). By inclusion oromission of SP in the vector, some of these systems (e.g. MalE, His Tag)can be adapted for periplasmic or cytosolic expression, respectively. Ingeneral, such vectors contain a highly specific protease cleavage sitefor downstream purification of the product. Fusions functional in bothdomains, e.g. MalE and secreted domain, can be obtained. This, however,is dependent on the nature of the protein. The carrier domain mayinterfere with folding of the recombinant protein resulting in proteindegradation, insolubility of the protein due to membrane association orformation of insoluble inclusion bodies at higher concentrations.

3.3. Surface Display in E. coli

Insertion of epitopes into major OM proteins (OmpA, LamB, PhoE),flagella, fimbriae. These systems involve insertion of epitopes into apermissive site, i.e. surface loop within OM proteins or flagellar,fimbriae subunits, without affecting assembly of the membrane protein orsurface appendage. In general, there are severe size restrictions of theinsert (10-60 amino acids) to avoid effects on folding and assembly ofthe protein. There are reports of surface display of whole proteins bypreparing terminal fusions to part of the outer membrane protein. OmpAor of IgA protease. Using a Lpp- OmpA vector, complete enzymes have beenlocalised to the surface of E. coli offering the potential of surfacedisplay, but these constructs lead to disruption of the outer membranewith concomitant toxicity to the cell and leakage of periplasmiccontents. In addition, the fusion proteins follow the outer membraneprotein assembly pathway. This limits the maximum number of surfacemolecules and more importantly it is evident that completely foldedproteins possessing disulphide bonds cannot be assembled across theouter membrane by this route (Klauser, T., et al. (1990) EMBO Journal.9:1991-1999; Stathopoulos, C., et al. (1996) Applied Microbiology andBiotechnology. 45:112-119).

3.4. Extracellular Secretion.

There have been limited reports on extracellular secretion of unrelatedproteins by some of the above mentioned secretion pathways. The Hly TypeI secretion pathway has been adapted to delivery of heterologousantigens (Gentschev, I, et al. (1996) Gene 179:133-140). Althoughapparently successful, this system delivers proteins directly from thecytosol and would preclude any protein which require exposure to theperiplasmic space for correct folding, e.g. disulphide bond formation.

It is summarised below some of the serious drawbacks associated withrecombinant protein expression:

(i) Periplasmic expression systems: Many heterologous polypeptidesexpressed in E. coli are either degraded or form aggregates andinclusion bodies as a result of incorrect folding. This may occurdespite targeting of the protein to a preferred location, i.e. thecytosol (with a more reducing environment) or the periplasm (with a moreoxidising environment and specific chaperones involved in folding).Employment of a signal sequence to proteins targeted to the periplasmresults in varying degrees of efficiency of precursor processing,completion of translocation and correct folding. Some incorrectly foldedproteins remain associated with the inner membrane and induce toxicity.In addition, they are extensively degraded resulting in a poor yield.Others accumulate in a non-native conformation as insoluble aggregates.Systems employing fusion proteins are available. These may to somedegree enhance solubility of some recombinant proteins but others remaininsoluble due to incomplete folding of the heterologous domain. A systemleading to stimulation of the early folding event followingtranslocation across the inner membrane would clearly enable periplasmicexpression of many heterologous polypeptides which have thus far eludedsuccessful expression in E. coli.

(ii) Surface localisation in Gram negative bacteria: Generally, there isa strict limitation in the size of epitopes which can be expressed atthe cell surface using proven surface expression vectors. Systems thatpermit surface expression of whole domains or proteins by fusion them toan outer membrane protein lead to membrane permeabilisation, periplasmicleakage and toxicity. In addition, there are limitations on the extentto which proteins can be folded if they are to be exported by thispathway. Finally, as these systems all use integral membrane proteins,they are limited with respect to the maximum expression level and wouldbe very laborious to purify.

SUMMARY OF THE INVENTION

The present invention provides bacterial strains for secretion ofsoluble biologically active recombinant heterologous proteins intoperiplasm or on a surface/into a cultivation medium of bacteria. Theinvention exploits the secretion system of Gram-negative bacteriaincluding periplasmic chaperones and usher/secretin proteins. Foraccomplishing the aim, the bacterial strains simultaneously express thefusion protein (signal peptide)-(mature heterologous protein)-(subunitof a bacterial surface structure, Caf1), periplasmic chaperone specificfor the subunit, and outer membrane usher/secretin protein specific forthe subunit. Secretion of fusion proteins: (signal peptide ofCaf1)-(mature human IL-1β)-(mature Caf1), (signal peptide ofCaf1)-(mature human GM-CSF)-(mature Caf1), and (signal peptide ofCaf1)-(mature human IL-1ra)-(mature Caf1) that were expressed inEscherichia coli simultaneously with the periplasmic chaperone Caf1M andthe usher/secretin protein Caf1A are examples of the use of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Extracellular secretion systems in Gram negative bacteria. Majorcomponents include: (i) Type I (e.g. HlyA)−inner membrane (IM) ABCtransporter (Hly B and D)+an outer membrane (OM) protein (Tol C), noperiplasmic intermediate detected; (ii) Type II (e.g. pullulanase)−Secsystem for translocation across IM; translocation across OM−14 pul geneproducts including an OM secretin (PulC , S), eight IM pul products (PulC, E,F, K-O) and four prepilin-like products (Pul G-J), evidencesupports periplasmic intermediate; (iii) Type II (e.g. Yops)−24 Yscproteins including an OM secretin (YscC) and IM ATPase (YscN), specificSyc cytosolic chaperones, and Yop B and D required for delivery toeukaryotic cell (iv) Type IV—newly classified group including systemsinvolved in DNA transfer to plant or other bacterial cells (e.g. T-DNAof Agrobacterium tumefaciens, 11 virB genes, unlikely to go viaperiplasmic intermediate) and pertussis toxin export (9 ptl genes,periplasmic assembly of toxin likely). (v) OM helper (e.g. IgAprotease)—Sec system for translocation across IM; no specific accessoryproteins; uses a ‘helper domain’ within the protein which presumablyfollows the OM protein assembly pathway and folds into the OM exposingthe secreted domain on the surface; (vi) Specific chaperone mediatedassembly (e.g.Pap pili)−Sec system for translocation across IM;translocation across OM−periplasmic chaperone (PapD) which specificallyrecognises pilin subunits, OM usher/secretin protein (PapC) homologousto PulC and YscC of TypeII and TypeIII pathways, respectively.

FIG. 2. Construction of hybrid genes coding for ^(s.p.)Caf1-hIL-1βfusion proteins (SEQ ID NOS: 20-25).

FIG. 3. Expression of ^(s.p.)Caf1-hIL-1β fusion proteins. A. CoomassieBlue-stained SDS-PAGE of soluble (lanes 2-5) and insoluble (lanes 6-9)proteins from E. coli cells transformed with pKKmod (lanes 2, 6),pKKmod/^(s.p.)Caf1-hIL-1β (lanes 3, 7), pKKmod/^(s.p.)Caf1(−2)hIL-1β(lanes 4, 8), and pKKmod/^(s.p.)Caf1(+3)hIL-1β (lanes 5, 9). hIL-1β wasloaded as a control (lanes 1, 10). B. Immunoblot of the same gelanalysed with anti-hIL-1β rabbit polyclonal antibodies. Positions ofunprocessed (I) and processed (II) hIL-1β are shown by arrows.

FIG. 4. Secretion of ^(s.p.)Caf1-hIL-1β fusion proteins. Immunoblots ofperiplasmic (A) and soluble cytoplasmic (13) proteins from^(s.p.)Caf1(+3)hIL-1β (lane 1), ^(s.p.)Caf1(−2)hIL-1β (lane 2), and^(s.p.)Caf1-hIL-1β (lane 3) expression strains. Corresponding proteinsfrom cells harbouring pKKmod were used for comparison (lane 4). hIL-1βwas loaded as a control (lane 5). Proteins were analysed withanti-hIL-1β rabbit polyclonal antibodies. Positions of unprocessed (I)and processed (II) hIL-1β are shown by arrows.

FIG. 5. Trypsin digestion of permeabilized cells. Insoluble proteins(lane 1-2) and soluble proteins (lane 3-4) were obtained from^(s.p.)Caf1(−2)hIL-1β expression cells before (lane 1,3) and after (lane2,4) trypsin treatment.

FIG. 6. Construction of pCIC plasmid coding for the^(s.p.)Caf1(−2)-hIL-1β-Caf1 fusion protein.

FIG. 7. The N-terminal sequence of mature CIC. After partialpurification of periplasmic fractions on a DEAE-Sepharose CL-6B column(Pharmacia, Sweden) proteins were separated by SDS-PAGE followed byblotting onto a PVDF-membrane (Amersham, UK). The desired bands wereexcised and placed onto a polybrene coated and precycled glass fiberfilter. Amino acid sequence analyses were performed with an AppliedBiosystems model 477A protein sequencer equipped with on-line AppliedBiosystems model 120A phenylthiohydantoin amino acid analyser.

FIG. 8. Caf1M facilitates expression of ^(s.p.)Caf1(−2)-hIL-1β-Caf1(CIC). A. Coomassie Blue-stained SDS-PAGE of periplasmic fraction fromcells transformed with: pCIC (lane 1), pFMA (lane 2), pMA-CIC (lane 3),pA-CIC (lane 4), pM-CIC (lane 5), pMA-PrCIC (lane 6), pA-PrCIC(lane 7),pM-PrCIC(lane 8), pCIC, pCaf1M (lane 9), pCIC, and pCaf1MA (lane 10). B.Immunoblot of the same gel analysed with anti-hIL-1β rabbit polyclonalantibodies.

FIG. 9. Graphical representation of plasmids constructed for operon-likeco-expression experiments with Caf1M, Caf1A, and CIC. pFMA and pCIC aregiven for comparison.

FIG. 10. Detection of CIC in periplasm with monoclonal anti-ILantibodies using ELISA. Detection of CIC in dilutions of a periplasmaliquot from control cells carrying pTrc99 with plasmid (triangles),from cells carrying pCIC (circles) and from cells carrying both pCIC andpCaf1MA (squares).

FIG. 11. Construction of pFGMF1 and pFRF275 plasmids coding for thescaf-GMCSF-Caf1 and scaf-IL1ra-Caf1fusion protein.

FIG. 12. Fractionation of proteins expressed in JM101 cells withplasmids pFGMF1 (lanes 3,4,7,8), pFRF275 (lanes 1,2,5,6), and pCaf1M(lanes 2,4,6,8). After induction cells were precipitated, resuspended inbuffer with lysozyme and incubated on ice for 1 hour followed bysonication for 1 min. Soluble and insoluble proteins were separated bycentrifugation. Lanes 1-4—insoluble fraction, lanes 5-8—solublefraction.

FIG. 13. Expression of scaf1-GMCSF-Caf1 fusion gene. A. SDS-PAGEanalysis of periplasmic proteins obtained from cells harboring pFGMF1(lane 1), pFGMF1 and pCaf1M (lane 2), and pFGM13 (lane 3). B. Proteinimmonoblot analyzed with anti-GMCSF rabbit polyclonal antibodies. Lanes1,2—total cell proteins, lanes 3-5—periplasmic proteins. Proteinfractions were obtained from cells harboring pFGMF1 (lane 1,4), pFGMF1and pCaf1M (lane 2,3), and pFGM13 (lane 5). C. lmmonoblot of periplasmicproteins analyzed with anti-Caf1 rabbit polyclonal antibodies. Cellsproduced GMCSF-Caf1 fusion protein without (lane 1) or with (lane 2) theCaf1M chaperon.

FIG. 14. Expression of scaf1-IL1ra-Caf1 fusion gene. A. SDS-PAGE ofperiplasmic proteins obtained from cells harbouring pFRF275 alone(lane 1) or with pCaf1M (lane 2). B. Immunoblot of the same gel analysedby anti-IL1ra goat polyclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, secretion of proteins bygram-negative bacteria can be accomplished with adhesins viachaperone/usher pathway. The periplasmic chaperones of the system:

-   -   mediate the partioning of nascently translocated subunits out of        the inner membrane and into the periplasm;    -   help in the folding of nascent subunits into a native        conformation;    -   protect the subunits from a proteolytic degradation.

The periplasm of wild type strains of bacteria contains oxidoreductasesand additional chaperones (disulphide bond isomerase, DsbA and C,peptidyl prolyl cis-transisomerase, RotA, SurA and FkpA) which areessential to the correct folding of proteins.

The caf operon is the simplest operon in comparison to the size of theoperons dedicated to most of the other extracellular secretion systems(Karlyshev, A. V. et al. (1994) in Biological Membranes: Structure,Biogenesis and Dynamic. NATO-ASI Series, vol. H-82, Op den Kamp, J. A.F., ed., pp. 321-330, Springer-Verlag, Berlin). It comprises of caf1Rencoding a transcription regulator, the structural gene for the Caf1polypeptide (Galyov, E. E. et al. (1990) FEBS Lett. 277, 230-232) andtwo genes encoding products for specific secretion of Caf1—the Caf1Mperiplasmic chaperone (Galyov, E. E. et al. (1991) FEBS Lett. 286.79-82)and Caf1A outer membrane protein (Karlyshev, A. V. et al. (1992) FEBSLett. 297, 77-80).

Cal1 polypeptide is synthesised with a 20 aa cleavable SP. Followingtranslocation across the inner membrane (presumably via the secpathway), Caf1M binds to mature Caf1 and protects it from proteolyticdegradation by assisting its folding and also its release from the innermembrane (Zav'yalov, V. et al. (1997) Biochem. J., 324, 571-578; ChapmanD. et al. (1999) J. Bacteriology, in press). Interaction of subunit withCaf1M chaperone then stimulates a signal for interaction with the OMprotein Caf1A and Caf1A translocates Caf1 to the cell surface. Surfaceassembled Caf1 forms an amorphous capsule-like structure on the surfaceof the bacteria. This surface structure can be readily recovered withthe bacterial cells and washed off the surface to give a relatively purepreparation of the Caf1 protein. Caf1M chaperone and Caf1A usher arespecific for Caf1 subunit and not suitable for a secretion ofheterologous proteins from this point of view. However, surprisingly itwas found in the present invention that the fusions of three differentheterologous proteins with the Caf1 subunit are secreted in solublebiologically active form and protected from proteolytic degradation whenexpressed simultaneously with the Caf1M chaperone.

According to the present invention Caf system can be applied for:

-   -   production of recombinant heterologous proteins in the periplasm        of bacteria in a soluble, biologically active conformation;    -   surface display of whole recombinant heterologous proteins on        Gram-negative bacteria—for obtaining heterologous live        attenuated vaccines, construction whole cell ligands, whole cell        biocatalysts, purificaton of recombinant heterologous protein        from cell surface by washing fusion protein out of the cell        surface, or by proteolytic cleavage of fusion protein;    -   surface display of heterologous amino acid sequences (epitopes)        within Caf1 subunit, and use as ligand, vaccine candidate etc.        Potential Applications of the Caf System:

-   1. Caf1SP-export across the IM: As with many other secretory    proteins the Caf1 signal sequence can be used directly for secretion    of proteins across the bacterial inner membrane. It contains no    positively charged residues at its C-terminus that might interfere    with export and can therefore be used directly. As the secreted    protein would follow the Sec pathway to the periplasm, the    efficiency of export of the heterologous protein would also depend    on the nature of the protein to be secreted. In particular,    efficiency of secretion would depend on the N-terminus of the mature    polypeptide, absence of long potential anchor sequences within the    secreted protein, and absence of protein folding prior to export.    The first two features can be addressed by altering the relevant    sequences. ‘Premature’ folding is more difficult to control although    excess of cytosolic SecB, GroEL, DnaK chaperone may delay folding in    some instances. Following translocation across the IM and SP    cleavage, release of the heterologous protein from the IM and    folding of the protein would again be dependent on the nature of the    heterologous protein itself.

-   2. Caf1M chaperoning in the periplasm: The caf system has the added    advantage that it includes a periplasmic chaperone which    specifically binds to the Caf1 subunit. Caf1M prevents degradation    of the Caf1 subunit, probably via enhanced folding and release from    the IM. Chaperone recognition is via the C-terminus of the Caf1    subunit although interaction with other parts of the Caf1 subunit    may also be required for high affinity binding. Hence, fusion of the    Caf1 subunit C-terminally to the protein destined for release may    stimulate folding of the heterologous protein, aid release of the    protein from the IM, increase its solubility and prevent aggregation    (formation of periplasmic inclusion bodies) and proteolytic    degradation.

-   3. Caf1A—export of heterologous proteins extracellularly across the    outer membrane. Caf1M chaperone also targets Caf1 to the OM secretin    Caf1A. Following interaction of the complex with Caf1A secretin, the    Caf1 is translocated across the outer membrane and forms a large    polymeric structure on the cell surface-anchored via Caf1A. For the    case that the anchoring is disfavored a proper mutant of Caf1 can be    prepared. Thus, inclusion of Caf1A in the expression vector would    permit targeting of the recombinant protein to the Caf1A secretin.

-   4. Hybrid proteins exported by this system could be proteolytically    cleaved to remove Caf1 by introduction of an appropriate cleavage    site between Caf1 and the protein of interest. For proteins arrayed    on the cell surface of E. coli this provides an enormously efficient    method of purification from isolated cells. Cell surface localised    hybrid could also be used for vaccine preparation, ligand binding or    as whole cell biocatalyst. Proteins secreted from the cell or    recovered from the periplasm would be already in a highly purified    form. All proteins secreted by this system would be exposed to the    periplasm, offering the milieu for correct folding of secreted    proteins. Using this system, the probability of folding into a    functional protein/domain would be further increased for some fusion    proteins due to interaction with the Caf1M chaperone.

In addition to secretion of whole proteins or domains as an N-terminalfusion to the Caf1 subunit (or possibly smaller domain thereof), it willundoubtedly also prove to be the case that short epitopes can beinserted into permissive sites within the Caf1 subunit permittingsurface display of epitopes. Similarly, short peptides can be insertedinto the FGL site of the Caf1M chaperone.

Summary of Potential Advantages of the Caf System as Applied toHeterologous Secretion n Gram Negative Bacteria:

-   -   despite the fact that protein hIL-1β is insoluble in the        periplasm and a fusion hIL-1β-Caf1 is also insoluble,        co-expression with the Caf1M chaperone surprisingly led to a        soluble product. Hence, the Caf1M chaperone catalyses folding,        not only of the carrier Caf1 domain, but also of the IL-1β        domain, resulting in release from the membrane and formation of        a processed, soluble product. Because of correct folding the        heterologous fusion protein is not degraded and accumulates in        high levels. I.e. Caf1M can stimulate folding, membrane release        and solubility of heterologous polypeptides expressed as Caf1        fusion proteins:    -   while available systems of surface display of whole proteins        in E. coli result in problems of loss of integrity of the outer        membrane and have limitations as to the extent of folding of the        exported protein, the caf system has not these limitations. The        fusion protein is anchored to the cell surface or released as a        soluble protein without disrupting the membrane. Similarly,        assembly does not follow the folding pathway of outer membrane        proteins and a fully folded fusion protein is exported;    -   option to readily express high levels of recombinant protein in        periplasm (Caf1M alone) or cell surface/media (Caf1M+Caf1) using        the same system;    -   ready purification from the cell surface by proteolytic cleavage        or recovery in high levels from periplasmic fractions (whichever        preferred);    -   exquisitely simple compared to other potential extracellular        secretion vectors-employing only a periplasmic chaperone (Caf1M)        and outer membrane protein (Caf1A);    -   ability to secrete extracellularly a whole protein or part        thereof    -   transient or permanent periplasmic localisation of fusion        protein permits correct folding and oxidation of secretory        proteins.

The invention is further illustrated here below with specificnon-limiting examples. The examples describe the secretion of the fusionproteins (signal peptide of Caf1)-(mature human IL-1β)-(mature Caf1),(signal peptide of Caf1)-(mature GM-CSF)-(mature human Caf1) and (signalpeptide of Caf1)-(mature human IL-1ra)-(mature Caf1) expressed inEscherichia coli simultaneously with the periplasmic chaperone Caf1M andthe usher/secretin protein Caf1A as the examples of the use of thesystem. Even though Examples describe secretion of 3 proteins it isobvious that any other protein can be secreted using this system. Inaddition, although E. coli is used as the host microbe containing theexpression system in these examples, it is obvious that also other cellshaving proper periplasmic space for accommodating the caf expressionsystem can be used.

EXAMPLE I Fusion or hIL-1β with the Signal Peptide of the Caf1 Protein

The Caf1 signal peptide (^(s.p.)Caf1) was fused with hIL-1β in threevariants (FIG. 2). 1. ^(s.p.)Caf1-hIL-1β was the straight fusion wherethe last amino acid of the Caf1 signal peptide was joint to the firstamino acid of hIL-1β. 2. ^(s.p.)Caf1(−2)-hIL-1β was the straight fusionwith a mutation in the Caf1 signal peptide Asn(−2)Asp. 3.^(s.p.)Caf1(+3)hIL-1β was the fusion containing in the joint regionthree N-terminal amino acids from mature Caf1 to preserve the naturalprocessing site.

Here and thereafter DNA manipulations and transformation of E. coli wereaccomplished according to Maniatis, T., et al. (1989) Molecular Cloning:A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, ColdSpring Harbor. Restriction enzymes, mung-bean nuclease, and T4 DNAligase were purchased from Promega (USA). Taq DNA polymerase (Hytest,Finland) was used for polymerase chain reaction (PCR) experiments.Nucleotide sequencing was carried out using the TaqTrack sequencing kit(Promega, USA). Elution of DNA fragments from agarose gels was performedwith the USBClean kit (USB, USA). The following oligonucleotides wereused in EXAMPLES 1 and 2 (SEQ ID NOS: 1-13):

-   CAF-RI 5′-GGGAATTCAGAGGTAATATATGAAAAAAATC-3′-   IL-PST 5′-CCGCCTGCAGATGCGGCACCTGTACGATCACTG-3′-   CAF-PST 5′-CCGCCTGCAGTTGCAATAGTTCCAAATA-3′-   IL-Primer 5′-AGAACACCACTTGTTGCTCC-3′-   Blunt 5′-TGGAACTATFGCAACTGCAAATGCGGCACCTGTACGA-3′-   3AA 5′-GCAACTGCAAATGCGGCAGATTTAGCACCTGTACGATC-ACTG-3′-   IL-BamHI 5′-ACCGGATCCACCTCCACCAGATCCACCTCCGGAAGACACA AATTGCATGG-3′-   BamHI-5′-GGTGGATCCGGTGGTGGTGGATCTGCAGAJTTAACTGCA-   Caf AGCAC-3′-   Caf-SalI 5′-GCCAAGCTTGTCGACGAGGGTTAGGCTCAAAGT-3′-   SBEKP-1 5′-TCGACAGATCTCGAATTCCGGTACCGGCTGCA-3′-   SBEKP-2 3′-GTCTAGAGCTTAAGGCCATGGCCG-5′-   STOP 5′-GATCATTAATTAAT-3′-   TRC 5′-CCAGATCTGGCAAATATTCTGAAATG-3′

The genetic constructs coding three fusion proteins ^(s.p.)Caf1-hIL-1βwere made according to the scheme in FIG. 2. The EcoRI-PstI fragment(about 110 bp) coding the Caf1 5′-untranslated region and N-terminalpart of Caf1 signal peptide with the mutation Asn(−2)Asp was obtains byPCR using CAF-RI and IL-Pst oligonucleotides and the template plasmidpKM4 (Karlyshev, A. V., et at. (1992) FEBS Letters 297, 77-80) followedby digestion of PCR1 product with EcoRI and PsI. The PstI-HindIIIfragment coding for the C-terminal part of Caf1 signal peptide joint tothe N-terminus of hIL-β was obtained by PCR where CAF-PST and IL-Primeroligonucleotides and the template plasmid pPR-TGATG-hIL-1β-tsr (Mashko,S. V., et al. (1991) Gene 97, 259-266) were used. The PCR2 product wasdigested with PstI and HindIII. The two fragments were ligated togetherwith a vector fragment pUC19/EcoRI-HindIII. The nucleotide structure ofthe resulted EcoRI-HindIII insert was verified by DNA sequencing. TheEcoRI-HindIII fragment obtained as described and the HindIII-BamHIfragment isolated from pPR-TGATG-hIL-1β-tsr were ligated together withthe EcoRI-BamHI vector fragment of pUCΔ19 ΔHindIII (a derivative ofpUC19 where the HindIII site was deleted by filling of sticky endsfollowed ban blunt-end ligation). The EcoRI-BamHI fragment thus obtainedencoded the ^(s.p.)Caf1(−2)hIL-β fusion protein. The point mutation G toA converting the ^(s.p.)Caf1(−2)hIL-1β gene into the Caf1-hIL-1β genewas made by means of two-step PCR procedure (Landt, O., Grunert. H.-P.and Hahn, U. (1990) Gene 96, 125-128) using the BLUNT oligonucleotideand two flanking primers (M13 Sequence Primer and IL-Primer) withpUC19ΔHindIII/^(s.p.)Caf1(−2)hIL-1β plasmid as a template. In the firststep, an intermediate PCR product was obtained from the mutagenic BLUNToligonucleotide and the M13 Sequence Primer. After purification from anagarose gel, the intermediate PCR product was used with the IL-Primer inthe second PCR step. The latter PCR product was digested with EcoRI andHindIII followed by ligation into the vector fragment made byrestriction of the pUC19ΔHindIII/^(s.p.)Caf1(−2)hIL-1β plasmid with thesame enzymes. Thus, the EcoRI-HindIII fragment providing the Asn(−2)Aspmutation in the ^(s.p.)Caf1(−2)hIL-1β fusion protein was replaced by thecorresponding fragment coding the natural Caf1 signal peptide, and the^(s.p.)Caf1-hIL-1β gene was obtained. The ^(s.p.)Caf1(+3)hIL-1β gene wasconstructed in a similar way using the 3AA oligonucleotide as amutagenic primer and pUC19ΔHindIII/^(s.p.)Caf1-hIL-β plasmid as atemplate. In all three hybrid genes the EcoRI-HindIII fragments weresequenced to prove correct nucleotide structures.

EcoRI-BamHI fragments coding for the fusion proteins were excised fromplasmids described above and were transferred into pKKmod (a derivativeof pKK 223-3 (Pharmacia) created by deletion of BamHI- and SalI-sites inthe tet gene region and by replacement of the pKK 223-3 polylinker withthe pUC18 polylinker). The expression plasmidspKKmod/^(s.p.)Caf1(−2)hIL-1β, pKKmod/^(s.p.)Caf1-hIL-1β andpKKmod/^(s.p.)Caf1(+3)hIL-1β were thus obtained, and E. coli JM105 [F′traD36, lacl⁹Δ(lacZ)M15, proA⁺B⁺/thi. rpsL(Str⁺), endA. sbcB. sbcC,hsdR4(r_(k) ⁻m_(k) ⁺), Δ(lac-proAB)] (NE BioLabs) were transformed withthese plasmids.

Expression and processing of ^(s.p.)Caf1-hIL-1β, ^(s.p.)Caf1(−2)hIL-β,and ^(s.p.)Caf1(+3)hIL-1β fusion proteins were monitored by SDS-PAGEelectrophoresis and immunoblotting. The recombinant E. coli strains weregrown to about 0.5 AU at 600 nm, IPTG (0.5 mM) was added and cells weregrown further. Soluble and insoluble proteins were analysed as separatefractions obtained as follows. After certain time intervals, samples ofthe growth culture with fixed amount of cells were withdrawn cells wereprecipitated and washed in 25 mM Tris-Cl, pH 7.5/100 mM NaCl/1 mM EDTA.Reprecipitated cells were suspended in 50 mM H₃PO₄-Tris, pH 6.8 andsonicated using Labsonic U Generator (B. Braun Diessel Biotech). Thesonicated samples were centrifuged at 14,000 g for 10 min. Supernatantcontained soluble proteins. Insoluble proteins were extracted from thepellet with the SDS-PAGE sample buffer containing 2% SDS and 5%β-mercaptoethanol.

Here and thereafter 10-15% SDS-PAGE of the proteins from both solubleand insoluble fractions was performed according to the Laemmli procedurein Mini-PROTEAN II apparatus (BIO-RAD). Proteins were transferred to aHybond-C membrane (Amersham) by electroblotting in mini-Trans-BlotElectrophoretic Transfer Cell (BIO-RAD) followed by immunodetection.Results of immunoblots were revealed with the ECL kit (Amersham).Polyclonal rabbit antibodies to hIL-1β (Calbiochem) and monospecificantibodies to Caf1 were used for visualisation of IL-1β andCaf1-containing fusion proteins. Rabbit polyclonal antibodies to Caf1Mand mouse policlonal antibodies to Caf1A were used for detection Caf1Mand Caf1A, correspondingly. The binding of the primary antibodies werevisualised by rabbit (Calbiochem) and mouse (Amersham) conjugate.

High expression was observed for all three fusion proteins (about 20-30%of total cell protein). However, in all three recombinant strainsfractions of soluble proteins did not contain significant amounts ofrecombinant proteins (FIG. 3A and B, lanes 1-4). The recombinant hIL-1βprotein was discovered in fractions of insoluble cell proteins (FIG. 3Aand B, lanes 6-9). An additional hIL-β containing protein was clearlyseen in all three constructs. Most probably, it was a product of ratherspecific proteolysis of unprocessed fusion protein accumulated incytoplasm.

The straight fusion appeared not to be processed (FIG. 3A and B, lanes7), while in the other two products the removal of Caf1 signal peptidesfrom hIL-1β moieties achieved considerable levels (FIG. 3A and B, lanes8,9).

To elucidate secretion of processed recombinant hIL-1β proteins,periplasmic proteins were separated from cytoplasmic proteins by osmoticshock procedure as follows. Cells precipitated from 10 ml of growthmedium were suspended in 200 μl 20% (w/v) sucrose/0.3 M Tris-HCl, pH8.0/0.5 MM EDTA and kept at room temperature for 10 min. Sucrose-treatedcells obtained by centrifugation were suspended in ice-cold 10 MMMgCI₂/0.1 mM PMSF and incubated in ice water for 10 min. Aftercentrifugation, periplasmic proteins were recovered in supernatantfractions. The pellets were suspended in 50 mM H₃PO₄-Tris, pH 6.8 andsonicated followed by centrifugation at 14,000 g for 10 min. Thesesupernatants contained soluble cytoplasmic proteins. The activity of thecytoplasmic enzyme, glucose 6-phosphate dehydrogenase, was checked as acontrol of the purity of the periplasmic fraction (Naglak T. J. et al.1990, 12, 603-611). The obtained samples of the periplasm fraction didnot show more than 0.25% of the cytoplasmic glucose 6-phosphatedehydrogenase activity.

Western blot analysis of periplasmic and soluble cytoplasmic proteins(FIG. 4) has shown that only minor parts of the processed fusionproteins were found in a soluble form in periplasms of^(s.p.)Caf1(−2)hIL-β and ^(s.p.)Caf1(+3)hIL-1β expression strains (FIG.4A, lanes 1,2). No processed protein was detected in periplasm of^(s.p.)Caf1-hIL-1β expression strain (FIG. 4A, lane 3). Processed hIL-1βproteins were absent in cytoplasmic fractions (FIG. 4B).

The data obtained demonstrated that the ^(s.p.)Caf1(−2)hIL-1β and^(s.p.)Caf1(+3)hIL-1β fusion proteins were partly processed (in contrastto the Caf1-hIL-1β fusion protein). Processed products were secretedinto periplasm. However, the majority of the processed recombinanthIL-1β, accumulated in an insoluble form. Moreover, this insolubleprotein is hidden from liquid phase of the periplasm since it is notdigested during trypsin treatment of permeabilized cells (FIG. 5).Variation of growth temperature and concentration of the inductor didnot facilitate the Caf1 signal peptide removal and secretion of hIL-1β(data not shown).

EXAMPLE 2 Fusion of ^(s.p.)Caf1(−2)hIL-β with the Caf1 Protein Sequenceand Expression of Fused Protein in the Presence of Chaperone and UsherProteins

The CIC (^(s.p.) Caf-IL-1β-Caf) protein was designed where the^(s.p.)Caf1(−2)hIL-1β amino acid sequence (N-terminus of the fusion) waslinked to the Caf1 protein sequence (C-terminus of the fusion) through aspacer GlyGlyGlyGlySer repeated three times. The spacer was inserted tominimise possible conformational problems of the two proteins fused inCIC.

The pCIC expression vector was constructed according to the scheme onFIG. 6. The IL part of CIC was obtained by PCR using IL-Pst and IL-BamHIoligonucleotides as primers and the pUC19ΔHindIII/^(s.p.)Caf1(−2)hIL-1βplasmid as a template. The Caf1 part of CIC was obtained by PCR usingBamHI-Caf1 and Caf1-SalI oligonucleotides as promers and the pKM4plasmid as a template. PCR products were digested with restrictases asshown in FIG. 3 followed by triple ligation with thepUC19ΔHindIII/^(s.p.)Caf1(−2)hIL-β vector obtained by digestion withPstI and SalI. To produce pCIC, EcoRI-SalI fragment was excised frompUC19ΔHindIII/CIC plasmid and ligated into the EcoRI-SalI vectorobtained from pTrc99ΔNco plasmid (a derivative of pTrc99a (Pharmacia)created by digestion with NcoI followed by mung-bean nuclease treatmentand ligation of blunt ends).

After IPTG induction. E. coli JM105 cells harbouring pCIC produced theCIC fusion protein that was successfully processed. A precise removal ofthe signal peptide was proved by N-terminal sequencing of the solubleperiplasmic CIC (FIG. 7). However, only a minor part of the matureIL-1β-Caf1 protein was extracted by an osmotic shock procedure (FIG. 8B,lane 1). Main part of the protein was found in the membrane fraction(data not shown), similar to mature hIL-1β.

Expression/secretion of CIC in the presence of the chaperone (Caf1M) andthe usher (Caf1A) was studied in two systems. a) caf1m and Caf1a geneswere situated on a pACYC plasmid compatible with the pTrc99 derivativecoding for CIC. b) caf1m, caf1a and cic genes together formed anartificial operon.

In compatible plasmid experiments, E. coli JM105 cells were transformedsimultaneously with pCIC and pCaf1M (a plasmid carrying caf1m gene undertac promoter in a pACYC184 derivative, see EXAMPLE 3) to study influenceof the chaperone on secretion of hIL-1β fusion protein. Further,pCaf1MA, a Caf1M-Caf1A expression/secretion plasmid, was created asfollows. ApaLI-ApaLI fragment containing caf1m and caf1a genes under trcpromoter was excised from the pFMA plasmid (Chapman D., et al.Structural and functional significance of the FGL sequence of theperiplasmic chaperone, Caf1M, of Yersinia pestis” J. Bacteriology, inpress) and ligated into the vector obtained by digestion of pCaf1M withApaLI. E. coli JM105 cells were transformed simultaneously with pCIC andpCaf1MA to obtain co-expression of all three proteins.

Several plasmids were constructed to study expression and secretion ofCIC, Caf1M and Caf1A proteins produced from an artificial operon (FIG.9). To replace the caf1 gene with a SBEKP synthetic polylinker, apMA-link plasmid was obtained by triple ligation of a pFMA/PstI-SpeIvector, a SpeI-PstI fragment of pFMA, and SBEKP-1 and SBEKP-2oligonucleotides annealed together. A plasmid pM-link was obtained fromthe pMA-link plasmid by excision of a SalI-SalI fragment coding for theCaf1A protein followed by self-ligation of the vector. A pA-link plasmidwas obtained from the pMA-link plasmid by excision of a BamHI-BamHIfragment coding for the C-terminal part of Caf1M protein. To interruptthe Caf1M translation frame, the stop-codon was inserted by ligation ofa self-complementary STOP oligonucleotide into the BamHI site. Theinsertion of the STOP oligonucleotide resulted in the loss of BamHIsite. A fragment coding for the CIC protein was excised from pCIC withEcoRI and SalI, cloned in pBCSK⁺ (Stratagene, USA), and recovered withEcoRI and KpnI. To obtain pMA-CIC, pM-CIC, and pA-CIC plasmids, theEcoRI-KpnI fragment was cloned into corresponding sites of pMA-link,pM-link, and pA-link. A plasmid pM-PrCIC differed from pM-CIC by thepresence of additional trc-promoter before the CIC gene, and wasobtained as follows. A DNA fragment coding for the trc-promoter and the5′-region of the cic gene was obtained by PCR using TRC and CAF-Pstoligonucleotides as primers and pCIC as a template. The PCR product wasdigested with BglII and EcoRI followed by ligation of the BglII-EcoRIfragment coding for the trc-promoter into corresponding sites of pM-CIC,pMA-PrCIC and pA-PrCIC were obtained by ligation of the BglII-KpnIfragment from pM-PrCIC into corresponding sites of pMA-link and pA-link.E. coli NM522 [F′, proAB, lacl⁹Δ(lacZ)M15/supE, thi-I, Δ(lac-proAB),Δ(hsdSM-mcrB)5, (r_(k) ⁻m_(k) ⁺)] (Stratagene, USA) was used as a hoststrain for plasmids described here.

Similar results were obtained in both co-expression methods.Simultaneous expression of CIC and Caf1M significantly increased theconcentration of the mature CIC in the periplasm. The resultsdemonstrated that periplasmic molecular chaperone promoted a release ofthe secreted protein from the inner membrane as well as preventedprotein from degradation and unspecific aggregation.

In the presence of Caf1M a significant part of the mature CIC proteinwas detected in a periplasmic fraction (FIG. 8B, lanes 3,4,6,7,9,10).Elevated amount of secreted and soluble CIC co-expressed with Caf1Mdemonstrates that the periplasmic molecular chaperone promotes a releaseof the secreted protein from an inner membrane. The critical role of theCaf1 part in the CIC protein in this promotion was proved in a similarexperiment with a plasmid differed from pCIC by deletion of one basepair in the spacer. The deletion resulted in the frame shift of the Cafpart of the fused gene. No facilitation of hIL-1β secretion was observedwhen cells harboured the mutated plasmid (data not shown).

Caf1M prevented CIC from degradation. As shown in FIG. 8B, lane 1, themature CIC protein in the periplasmic fraction rapidly degraded into atruncated form with a molecular weight about 20-23 kDa. In the presenceof Caf1M the truncated form disappeared (FIG. 8B, lanes 3,4,6,7,10). Theamount of the degraded form correlated with the amount of Caf1M. Forexample, when Caf1M was expressed from pCaf1M at a low level thetruncated form was found (FIG. 8B, lane 9). However, the Caf1M amountwas sufficient to facilitate a release of the fusion protein from themembrane. The specific digestion of the mature CIC occurred at a site inthe Caf1 part of the fusion protein since the truncated form was welldetected by the IL antibodies but not detected by the Caf1 antibodies(data not shown). Most probably the cleavage was due to the action ofprotease DegP, which had been shown is induced by unfolded secretedcapsular or pilus subunits and cleaves them (Soto, G. E., et al. (1998)EMBO J., 17, 6155-6167).

The hIL-1β part of CIC secreted in the presence of Caf1M was correctlyfolded. The mature CIC protein was detected with monoclonal antibodiesto hIL-1β in ELISA (FIG. 10). ELISA was performed using monoclonal mouseantibodies to hIL-1β (HyTest, Finland) as it was described previously(Zav'yalov, V., et al. (1997) Biochem. J., 324, 571-578).

CIC was excreted onto a cell surface when expressed with both Caf1M andCaf1A. The presence of the molecular chaperone and the usher protein incells transformed with pMA-CIC or with pCIC and pCaf1MA together wasproved by immunoblotting with anti-Caf1M and anti-Caf1A antibodies (datanot shown). To prove the excretion of CIC onto a cell surface, cellagglutination experiments were performed with reticulocyte monoclonaldiagnosticum for detection of Yersinia pestis (Middle Asian ReaserchInstitute, USSR). The E. coli cells expressed CIC, Caf1M, Caf1A wereable to precipitate reticulocytes with surface bound monoclonal antibodyto Caf1 at the concentration about 10⁷ cells/ml. Moreover, according toELISA performed with anti-hIL-1β monoclonal antibodies, a small amountof hIL-1β was detected in cultural medium.

EXAMPLE 3 Co-expression of hGMCSF-Caf1 Fusion Protein and the Chaperone

The expression plasmid for the hGM-CSF-Caf1 fusion protein was based onpFGM13 described in: Petrovskaya L. E. et al. (1995) Russian Journal ofBio-organic Chemistry, 21, 785-791. The pFGM13 plasmid contained asynthetic gene coding for hGMCSF with Caf1 signal sequence (scaf1)cloned into HindIII and XbaI sites of pUC19. Transcription wascontrolled by the lac promoter and was inducible with IPTG. Translationof the scaf1-gmcsf gene in pFGM13 initiated at the first methioninecodon of lacZ gene and utilises its Shine-Dalgarno sequence. A primarystructure of the signal sequence (scaf1) encoded by this gene differedfrom wild-type one at its N-terminus, which contained seven extraresidues from β-galactosidase N-terminus.

The construction of the pFGMF1 plasmid coding for the gmcsf-caf1 gene isshown in FIG. 11. The scaf1-gmcsf gene of pFGM13 was modified for thefollowing cloning of the fragment from pCIC containing a spacer(4GlySer)₃ and the Caf1 coding region. The Kpn2I site was introduced at3′-terminus of the gmcsf gene by PCR using two primers(5′ATCGGAAATGTTCGACCTTCAAG (SEQ ID NO: 14) and5′ATTATTCCGGACTCCTGCACTGGTTCCCAGC) (SEQ ID NO: 15) and pFGM13 as atemplate. Pfu DNA Polymerase was used for a maximal fidelity. The PCRfragment was treated with Kpn2I followed by ligation into theEcoRV-SalGI large fragment of pFGM13 together with Kpn2I-SalGI fragmentfrom pCIC. The final plasmid (pFGMF1) contained the gene encoding ahybrid precursor consisting of the scaf1 signal sequence, GMCSF with 2N-terminal amino acid changes (Ala₂Pro₃ to Asp), a Ser(4GlySer)₃ spacer,and Caf1. The plasmid structure was confirmed by restriction analysisand sequencing of amplified regions.

Expression of the hybrid precursor gene in JM101 E. coli cellsharbouring pFGMF1 was induced with 0.2 mM IPTG after cell culturereached an optical density 0,5-0.8. The growth with IPTG continued for 3I Cells from 1 ml were collected by centrifugation and the pellet (totalcell protein sample) was analysed by SDS-PAGE electrophoresis. Afraction of periplasmic proteins was isolated by the cold osmotic shockprocedure (see EXAMPLE 1) from the cell pellet obtained from the rest ofthe culture.

SDS-PAGE analysis of the total cell protein sample from E. coli cellsharbouring pFGMF1 revealed the presence of a large amount of the fusionprotein with molecular mass corresponding to the hGMCSF-Caf1 fusionprotein (31 kDa). When the cells were destroyed by sonication thisprotein was localised in an insoluble fraction (FIG. 12, lane 3, 4).

Examination of periplasmic extracts by SDS-PAGE and Western blotting hasshown that some hGMCSF-Caf1 fusion was translocated to the periplasm.Its electrophoretic mobility was the same as of the insoluble protein.The fusion interacts with both anti-hGMCSF and anti-Caf1 polyclonalantibodies (FIG. 13B, lanes 1; 14C, lane 1). The protein with molecularweight about 18 kDa was also detected with anti-hGMCSF antibodies (FIG.13B, lane 4). We can presume that this protein is the product ofhGMCSF-Caf1 fusion proteolytic degradation.

To study influence of the Caf1M chaperone on expression of thegmcsf-caf1 gene, the caf1m gene was inserted into the pACYC-trx, avector with a low copy number and compatible with pBR322-based plasmids.PACYC-trx was pACYC184 derivative that contained trx gene under thecontrol of tac promoter. The Trx coding region, flanked with KpnI andAlw44I sites, was replaced by the DNA fragment coding for the Caf1Mchaperone as follows. The caf1m gene was amplified by PCR which includedPfu DNA Polymerase, two primers (GTTGTCGGTACCATTCCGTAAGGAGG (SEQ ID NO:16) and 5′-GTTAACGTGCACACAGGAACAGC) (SEQ ID NO: 17) and the pFS2 plasmid(Galyov EE et al. (1990) FEBS Letters, 277, 230-232). The PCR fragmentwas treated with KpnI and Alw44I and cloned into the KpnI-Alw44I largefragment of pACYC-trx. The plasmid obtained was designated as pCaf1M.

JM101 E. coli cells were transformed with both pFGMF1 and pCaf1Mplasmids followed by analysis of recombinant cell proteins as describedabove. Co-expression of the chaperone gene from the pCaf1M plasmid ledto a marked increase in the amount of the full-length hGMCSF-Caf1 fusionprotein in the periplasm (FIG. 13B, lane 3; 13C, lane 2). The amount of18 kDa protein, the product of hGMCSF-Caf1 fusion proteolyticdegradation, decreased. These results demonstrate that Caf1M promotesthe correct folding of hGMCSF-Caf1 and enhances the stability of thefusion protein.

EXAMPLE 4 Co-expression of hIL1ra-Caf1 Fusion Protein and the Chaperone

Based on pFGM13, the pFRA275 plasmid was obtained. In this plasmid the5′-terminal part of the mature hIL-1ra coding region containdAlaAspAsp-coding sequence instead of the first Arg codon in order toneutralise the N-terminal positive charge of hIL1ra. The lac promotercontrolled the expression of scaf-hil1ra gene in pFRA275.

The expression plasmid pFRF275 coding for the scaf-hil1ra-caf1 gene wasconstructed as shown in FIG. 11. At the 3′ terminus of the hil1ra genethe Kpn2I site was introduced by PCR of the pFRA275 plasmid with primers5′-GGAATCCATGGAGGGAAGAT (SEQ ID NO: 18) and5′-ATTATTCCGGACTCGTCCTCCTGAAAGTAG(SEQ ID NO: 19). The amplified fragmentwas cut with NcoI and Kpn2I and ligated with the HindIII-Kpn2I largefragment from pFGMF1 together with the HindIII-NcoI fragment frompFRA275. The resulting plasmid (pFRF275) contained a gene which encodeshybrid precursor consisting of the scaf signal sequence, hIl-1ra withamino acid changes mentioned above, a Ser(4GlySer)₃ spacer, and Caf1.Plasmid structure was confirmed by restriction analysis and sequencingof amplified regions.

Expression of the scqf-hil1ra-caf1 gene in JM101 E. coli cells wasanalysed as described in EXAMPLE 3. When cells were transformed withpFRF275 alone, the hIL1ra fusion protein accumulated mainly in aninsoluble form (35 kDa. FIG. 12, lane 1, 2). However, some part of thefusion protein was translocated to the periplasm, where it was subjectedto degradation (FIG. 14B, lane 1). When pFRF275 and pCaf1M (see EXAMPLE3) were simultaneously present in recombinant cells. Caf1M noticeablydiminished the amount of cleavage products (FIG. 14B, lane 2).

1. A Gram-negative bacterial strain simultaneously expressing a fusionprotein comprising a signal peptide of Caf1, a mature heterologousprotein, and a subunit of a bacterial surface structure, which is Caf1from Yersinia pestis, and a periplasmic chaperone Caf1M specific forsaid subunit for secretion of a recombinant heterologous protein intoperiplasm of bacteria.
 2. The bacterial strain according to claim 1,additionally expressing outer membrane usher or secretin protein Caf1Aspecific for said subunit, for the purpose of secretion of a solublerecombinant heterologous protein on an outer surface of the bacterium orinto cultivation medium of the bacterium.
 3. The bacterial strainaccording to claim 1 or 2, wherein the microbe is Escherichia coli.
 4. Amethod for producing a heterologous recombinant protein which issecreted into the periplasm of a bacterium, comprising cultivating aGram-negative bacterial strain simultaneously expressing a fusionprotein comprising a signal peptide of Caf1, a mature heterologousprotein, and a subunit of a bacterial surface structure which is Caf1from Yersinia pestis, and a periplasmic chaperone Caf1M specific forsaid subunit, whereby the recombinant heterologous protein produced issecreted into the periplasm of the bacterium.
 5. The method according toclaim 4, wherein the heterologous recombinant protein is selected fromthe group consisting of GMCSF, IL-1β, and IL-1 receptor antagonist.
 6. Amethod for producing a heterologous recombinant protein which issecreted onto the outer surface of a bacterium or into a cultivationmedium of a bacterium, comprising the steps of cultivating aGram-negative bacterial strain simultaneously expressing a fusionprotein comprising a signal peptide of Caf1, a mature heterologousprotein, and a subunit of a bacterial surface structure which is Caf1from Yersinia pestis, a periplasmic chaperone Caf1M specific for saidsubunit, and outer membrane usher or secretin protein CalfA specific forsaid subunit, whereby the recombinant heterologous protein produced issecreted onto the outer surface of the bacterium or into the cultivationmedium of the bacterium.